The ubiquitin‐specific protease 5 mediated deubiquitination of LSH links metabolic regulation of ferroptosis to hepatocellular carcinoma progression

Abstract Epigenetic regulators and posttranslational modifications of proteins play important roles in various kinds of cancer cell death, including ferroptosis, a non‐apoptotic form of cell death. However, the interplay of chromatin modifiers and deubiquitinase (DUB) in ferroptosis remains unclear. Here, we found that ubiquitin‐specific protease 5 (USP5) is regarded as a bona fide DUB of lymphoid‐specific helicase (LSH), a DNA methylation repressor, in hepatocellular carcinoma (HCC). Functional studies reveal that USP5 interacts with LSH and stabilizes LSH by a deubiquitylation activity‐dependent process. Furthermore, the USP5‐mediated deubiquitination of LSH facilitates the tumorigenesis of HCC by upregulating solute carrier family 7 member 11 (SLC7A11) to suppress ferroptosis of liver cancer cells. Moreover, the USP5 inhibitor degrasyn inhibits DUB activities of USP5 to LSH to suppress the progression of HCC. Additionally, USP5 and LSH are positively correlated and both are overexpressed and linked to poor prognosis in HCC patients. Together, our findings show that USP5 interacts with LSH directly and enhances LSH protein stability through deubiquitination, which, in turn, promotes the development of HCC by suppressing ferroptosis of liver cancer cells, suggesting that USP5 may be a potential therapeutic target for HCC.

with LSH directly and enhances LSH protein stability through deubiquitination, which, in turn, promotes the development of HCC by suppressing ferroptosis of liver cancer cells, suggesting that USP5 may be a potential therapeutic target for HCC.

INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the most common malignant tumor entities, ranking sixth in incidence and third in mortality worldwide. 1 Due to the high occurrence of metastasis and recurrence of HCC, limited efficacy of radiotherapy and chemotherapy for the majority of HCC patients, and surgical treatment limited to early tumor stages, the underlying mechanism of HCC is urgently needed to be determined for the effective treatment of HCC. [2][3][4][5] Ferroptosis, a metabolism-related and regulated cell death, predominantly brought on by intracellular iron catalytic activity and lipid peroxidation, is defined by intracellular lipid peroxide accumulation and redox imbalance. [6][7][8][9] Ferroptosis is involved in some degenerative diseases, various kidney diseases, liver diseases, brain injury, and stroke as a stress response. 7 Meanwhile, numerous studies show that the great susceptibility of cancer cells to ferroptosis presents a rare opportunity for treating various kinds of cancers, especially in HCC. 10,11 Ferroptosis is one of the potential mechanisms of sorafenib in the treatment of HCC through the cystine/glutamate antiporter SLC7A11. 12 Inhibition of regulators, such as retinoblastoma, nuclear factor erythroid 2-related factor 2 (NRF2), and metallothionein-1g (MT-1G) that suppress the sorafenib-induced ferroptosis, could improve the resistance of sorafenib, and the combination therapy of haloperidol with sorafenib also promotes the sorafenibinduced ferroptosis. 13 In addition, extracts and monomers from natural products, including Chinese herbs, provide new strategies for the induction of ferroptosis to inhibit HCC. 14 Moreover, all these ferroptosis-targeting drugs induce ferroptosis of HCC cells mainly through iron metabolism, system xc−/GSH/glutathione peroxidase 4 (GPX4), p53, lipid peroxidation, and p62-Keap1-Nrf2 pathway. 15 Epigenetics, which determines gene transcription and has a significant impact on cell fate and developmental processes, mainly includes DNA methylation, histone modifications, noncoding RNAs, and chromatin remodeling. 16,17 Cancer cells frequently disrupt preexisting epigenetic systems to reprogram gene expression in a way that promotes the growth of cancer. Recently, many research have shown that a variety of epigenetic regulators, including lncRNA, deubiquitinase (DUB), and selenium, play crucial roles in ferroptosis by modulating metabolic genes and intermediaries, which results in a change in the level of lipid peroxidation. 15 Lymphoid-specific helicase (LSH), often referred to as helicase, lymphoid specific or proliferation-associated SNF2-like gene (PASG), an sucrose non-fermenter (SNF2) chromatin remodeling enzyme, 18 plays a vital role in preserving DNA methylation during the growth of plants and mammals. [19][20][21][22] In addition, LSH is crucial in repairing DNA-damage to maintain the genome stability of somatic cells. 23 Our previous research studies have shown that LSH also functions as a critical ferroptosis inhibitor in the tumorigenesis of lung cancer and leukemia. [24][25][26] However, it is still unclear how LSH affects the ferroptosis of HCC. In addition, LSH is overexpressed in various cancers, such as melanoma, head-and-neck cancer, and prostate cancer, and its overexpression leads to poor prognosis in several cancers. 27 Thus, LSH is probably involved in the cancer progression. However, the mechanism of LSH overexpression in cancers remains elusive, only a few studies have been conducted on it. 28,29 Moreover, the high LSH expression in tumor cells indicates the presence of LSH stabilizing factor.
Posttranslational modifications (PTMs) of proteins, which include ubiquitination, acetylation, SUMOylation and phosphorylation, play essential roles in regulating nearly all kinds of biological processes. 30 Among them, ubiquitination, a common PTM reversible by reactions induced by DUBs, targets many oncogenes and tumor suppressor genes involved in the progression of cancers. 31,32 Ubiquitin-specific protease 5 (USP5), a member of the USP family, the largest family of DUBs enzymes, also known as ubiquitin isopeptidase T, is distinctive in that it can specifically identify and remove ubiquitin from the proximal end of the unanchored polyubiquitin chains. 33,34 Multiple cellular activities, including the repair of DNA double-strand breaks, inflammatory responses, and stress responses, are regulated by USP5. [35][36][37] In this study, we reported that USP5 is an inhibitor of ferroptosis of HCC cells that promotes the development of HCC through stabilizing LSH protein. We revealed a previously unappreciated regulatory mechanism of LSH and the significant inhibitory effect of degrasyn on the tumorigenesis of HCC. Our study may provide a novel antitumor therapy of HCC by targeting USP5.

USP5 interacts with LSH
To completely clarify its potential regulatory mechanism, LSH-interacting proteins were identified using mass spectrometry in 293T cells stably expressing LSH. 38 LSH was found to be associated with numerous proteins, including USP5 ( Figure 1A). Then the correlation analysis between USP5 and LSH in the Cancer Genome Atlas (TCGA) HCC samples from Expression Profiling Interactive Analysis (GEPIA) revealed that USP5 and LSH were positively associated ( Figure 1B). USP5 and LSH colocalized mainly in the nucleus of HepG2 and Hep3B cells according to the immunofluorescence assay ( Figure 1C). To further demonstrate this interaction between USP5 and LSH, coimmunoprecipitation experiments were performed, and the results showed that USP5 could coimmunoprecipitate with LSH endogenously in HepG2 cells and exogenously in 293T cells ( Figure 1D,E). Moreover, deletion analyses were generated to map the domains of USP5 and LSH responsible for the interaction, and we discovered that the interaction between USP5 and LSH depended on the Nterminus of LSH ( Figure 1F,G). In conclusion, these data show that USP5 can directly interact with LSH.

USP5 stabilizes the LSH protein through deubiquitination
To further investigate the relationship between USP5 and LSH, we first examined the levels of protein expression of USP5 and LSH in normal liver L-02 cells and several liver cancer cells, and the results showed that USP5 was significantly expressed and positively correlated with LSH in HCC cells (Figure 2A). Many studies had proved that C335 is the key catalytic residue of USP5. [39][40][41] In our study, USP5-WT overexpression elevated LSH expression in LM3 cells, but not USP5-C335A ( Figure 2B,C). In addition, a stable overexpression of USP5 in HepG2 and LM3 increased the protein expression of LSH ( Figure 2D), knockout of USP5 in Hep3B induced the reduction of LSH protein level ( Figure 2E), and the level of LSH did not change after transfecting USP5-C335A into 293T ( Figure 2F). Moreover, the mRNA level of LSH did not change ( Figure  S1A), and the change of the LSH protein expression due to USP5 overexpression or knockout could be rescued by proteasome inhibitor Z-Leu-Leu-Leu-al (MG132) 42 ( Figure 2G, Figure S1B), but not restored by lysosomal inhibitor chloroquine (CQ) 43 ( Figure S1C). Meanwhile, cycloheximide (CHX) 44 chase assays showed that the half-life of LSH was prolonged by the overexpression of USP5 and decreased in USP5-knockout cells ( Figure 2H,I, Figure S1D). Thus, we confirmed that USP5 inhibited LSH degradation to stabilize LSH protein through proteasome pathway. As USP5 is a DUB, we continued to test whether USP5-regulated LSH protein stability via deubiquitination. The DUB assays demonstrated that the ubiquitin level of LSH was decreased in USP5-overexpressed cells, but did not happen in USP5-C335A-overexpressed 293T cells, and knockout of USP5 increased the ubiquitination of endogenous LSH ( Figure 2J,K, Figure S1E-G). Furthermore, the experiments to detect which types of ubiquitin of LSH were deubiquitylated by USP5 showed that USP5 significantly cleaved the K11-linked ubiquitin chain and slightly removed the K63-linked ubiquitin chain from LSH ( Figure 2L). Taken together, we found that USP5 stabilized the LSH protein through DUB LSH.

Overexpression of USP5 inhibits ferroptosis
In our previous studies, we demonstrated that LSH inhibits ferroptosis in lung cancer and leukemia by reducing the intracellular concentrations of iron and lipid ROS. 24,26 However, the function of LSH in ferroptosis of HCC remains unclear. To address whether USP5 could regulate ferroptosis by stabilizing LSH in HCC, we first used gene set enrichment analysis to explore the effects of USP5 on ferroptosis in HCC, and the results revealed that "WikiPathways Ferroptosis," "reactive oxygen species pathway," and "iron ion transport" showed enrichment differences between high and low USP5 expression group ( Figure 3A). Then we treated LM3 cells and HepG2 cells with RSL-3, 45 and the results of morphological observations showed the percentage of dead cells induced by RSL-3 decreased after USP5 overexpression ( Figure 3B, Figure S2A). Moreover, the cell counting kit-8 (CCK8) analysis indicated that USP5 overexpression inhibited the RSL3-induced ferroptosis of LM3 cells and HepG2 cells ( Figure 3C, Figure S2B). Western blot to detect the ferroptosis-associated markers revealed that acylcoenzyme A synthetase long-chain family member 4 (ACSL4), one of the ferroptosis promoters, was decreased F I G U R E 1 Ubiquitin-specific protease 5 (USP5) interacts with lymphoid-specific helicase (LSH). (A) The particular interaction information between USP5 and LSH was shown by the mass spectrometry analysis of LSH-associated proteins in 293T cells. (B) Correlation analysis of mRNA level between USP5 and LSH in the Cancer Genome Atlas (TCGA), hepatocellular carcinoma (HCC) samples from Expression Profiling Interactive Analysis (GEPIA). (C) Immunofluorescence assay of the co-localization of USP5 and LSH in HepG2 and Hep3B cells. (D and E) Analysis of the endogenous or exogenous interaction between USP5 and LSH using coimmunoprecipitation (Co-IP) and immunoblotting (IB). HepG2 (D) and 293T (E) were immunoprecipitated followed by IB with anti-USP5 or anti-LSH antibody. (F and G) Structure of LSH and FLAG-LSH fragments, including LSH-CC (coiled coil), LSH-DEXD (DEAH box domain), and LSH-C-term (helicase C-terminal) (F). For interaction between LSH truncation mutants and full-length USP5, IP analysis was followed by an IB assay (G).  by USP5 overexpression. Meanwhile, solute carrier family 7 member 11 (SLC7A11), the cystine/glutamate antiporter, known as a key suppressor of ferroptosis, was increased by the overexpression of USP5 ( Figure 3D, Figure S2C). Both of the intracellular levels of lipid ROS and GSH were surrogate markers for ferroptosis. We found that after treating cells with RSL-3, the overexpression of USP5 increased GSH levels and decreased the lipid ROS levels in LM3 cells and HepG2 cells ( Figure 3E,F, Figure S2D,E). And the detection of ferroptosis-associated markers in xenograft tumor samples with USP5 overexpression also showed that ACSL4 decreased, and SLC7A11 increased ( Figure 3G). However, after treating HepG2 cells transfected with USP5-C335A with RSL-3, the ferroptosis level of HepG2 cells did not show an obvious change indicated by CCK8 and the detection of GSH and lipid ROS ( Figure 3H,I, Figure S2E).

Knockout of USP5 promotes ferroptosis
After treating Hep3B cells with RSL-3, we found that the percentage of dead cells induced by RSL-3 increased after the knockout of USP5 by morphological observation ( Figure 4A). Meanwhile, the knockout of USP5 promoted the ferroptosis level of Hep3B cells indicated by the CCK8 analysis ( Figure 4B). And the results of western blot showed that ACSL4 increased, and SLC7A11 decreased after the knockout of USP5 ( Figure 4C). Moreover, the detection of GSH and lipid ROS of Hep3B cells treated with RSL-3 delivered that knockout of USP5 could decrease the GSH level and increase the lipid ROS level of Hep3B cells ( Figure 4D,E). Furthermore, we observed that the promotions of ferroptosis induced by knockout of USP5 were rescued by overexpressing LSH in USP5-knockout Hep3B cells ( Figure 4F-I). Taking together, our results demonstrated that USP5 inhibited ferroptosis by stabilizing LSH protein to a certain extent in HCC.

USP5 promotes tumor growth dependent on stabilizing LSH
To investigate the effect of USP5 in HCC tumor growth and migration, USP5-overexpressed HepG2 and LM3 cells were established to perform the gain-of-function studies. We found that cell proliferation, cell migration, and colony formation ability of HepG2 ( Figure S3A-C) and LM3 cells ( Figure 5A-C, Figure S4A,B) increased upon the overexpression of USP5, indicating that USP5 promotes HCC growth and migration. However, the HepG2 cells transfected with USP5-C335A did not show the increased ability of proliferation and migration ( Figure 5D-F, Figure   S4C,D). To further confirm the oncogenic functions of USP5 on HCC in vivo, USP5-overexpressed LM3 cells were subcutaneously transplanted into nude mice. In this experiment, tumor size and weight were obviously increased in mice with USP5 overexpression ( Figure 5G, Figure  S4E). These findings suggested that the growth of HCC is promoted by USP5 overexpression. Additionally, the loss-of-function study demonstrated that USP5 deletion markedly reduced cell proliferation, migration, and colony formation in Hep3B cells ( Figure 5H-J, Figure S4F,G). And tumor growth was suppressed in nude mice receiving USP5 knockout transplants ( Figure 5K, Figure S4H). In order to further detect whether the oncogenic functions of USP5 on HCC worked through stabilizing LSH, we first transiently overexpressed LSH in Hep3B cells stably knockout of USP5. The results showed that LSH overexpression efficiently rescued the cell growth and migration arrests induced by the depletion of USP5 ( Figure 5L,M, Figure S4I,J). Then we transiently knockdown LSH in Hep3B cells overexpressing of USP5 to further prove the LSH-dependent oncogenic role of USP5, and we found that the knockdown of LSH expression could decrease the oncogenic function of USP5 ( Figure S4K-N). In summary, we demonstrated that USP5 promoted liver cancer cells proliferation dependent on stabilizing LSH.

Degrasyn promotes ferroptosis to inhibit tumor progression via targeting of USP5
We used degrasyn, a specific DUB inhibitor, to further confirm our findings. 46 First, we treated HepG2 cells with degrasyn at increasing concentrations, 47 and we found that both USP5 and LSH decreased with increasing inhibitor concentration ( Figure 6A). In addition, the reduction of LSH caused by degrasyn could be rescued by MG132 ( Figure 6B), but not CQ ( Figure S5A), and co-treatment of CHX and degrasyn decreased the level of LSH more quickly ( Figure 6C). Deubiquitination assay also indicated that ubiquitin of LSH increased in degrasyn-treated HepG2 cells ( Figure 6D). These results showed that degrasyn reduced USP5 to promote the ubiquitination of LSH. We also found that USP5 inhibitor degrasyn suppressed cell proliferation, cell migration, and colony formation ability of HepG2 cells ( Figure 6E-G, Figure S5B,C). Then, in order to prove that the promotion effect of USP5 on the development of liver cancer was a common phenomenon in HCC and the potential value of targeting USP5 in the treatment of HCC, we used the xenograft nude mice model composed of another live cell line HepG2 cells to further reveal that the treatment of degrasyn targeting USP5 resulted in a decrease of the HCC tumor growth (Figure 6H,  Figure S5D). Furthermore, we demonstrated that in HepG2 cells treated with RSL-3, degrasyn could target USP5 to suppress cell growth, increase lipid ROS, and decrease GSH level ( Figure 6I-K). Moreover, the dead cells induced by RSL-3 increased after the treatment of degrasyn through morphological observations ( Figure 6L).
Meanwhile, western blot to detect ferroptosis-associated markers of degrasyn-treated HepG2 cells ( Figure 6M) and xenograft tumor samples ( Figure 6N) showed that key markers of ferroptosis, SLC7A11, and GPX4 decreased obviously. Conclusively, all these results revealed that degrasyn could target USP5 to promote the ferroptosis in HCC.

USP5 is upregulated and associated with poor survival in HCC
Last, we analyzed the mRNA expression of USPs family to find that among these USPs, USP5 obviously increased in HCC samples compared with normal liver tissue ( Figure 7A, Figure S6A). Moreover, western blot for detecting the expression level of USP5 in six pairs of HCC tissues and para-tumor tissues further demonstrated that USP5 was significantly higher in tumor tissues ( Figure 7B). Moreover, immunohistochemistry (IHC) staining for USP5 and LSH showed that both of them were elevated in Moreover, the increased LSH regulates the expression of ferroptosis-related proteins. And the ability of USP5 to stabilize LSH can be inhibited by degrasyn. ***p < 0.001 and ****p < 0.0001. HCC, and the positive staining of USP5 was located in the nucleus and cytosol ( Figure 7C, Figure S6B). In addition, USP5 was positively correlated with tumor grade of TCGA HCC samples ( Figure 7D). We performed survival analysis in HCC patients, and the results showed that a poor prognosis was linked to the high expression of USP5 and LSH ( Figure S6C,D), and patients with high expression of both USP5 and LSH showed the worst prognosis ( Figure 7E).

DISCUSSION
The levels of chromatin remodeling enzyme LSH are elevated and associated with poor clinical outcomes in various cancers, and substantial evidence suggests that LSH promotes the malignant progression of tumor cells. 38,48 A deeper understanding of the upstream regulatory mechanism of LSH is important for the discovery of new target for the treatment of cancer. DUBs protect proteins from degradation by removing ubiquitin from substrates. 49 Here, we have identified USP5 as a bona fide regulator of LSH. First, USP5 interacts with LSH directly. Second, USP5 enhances LSH protein stabilization through deubiquitination, and this ability could be inhibited by degrasyn, an USP5 inhibitor. Finally, USP5 could inhibit ferroptosis through DUB LSH to promote tumor progression. Deubiquitination is relevant for the occurrence and development of tumorigenesis. 50,51 Many researches have proved that DUBs involve in the regulation of some cancerrelated pathways, such as Akt (protein kinase B), Wnt/βcatenin signaling, transforming growth factor-β, and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells). [52][53][54][55] Meanwhile, multiple studies suggest that the effects of DUBs on ferroptosis-related proteins could be a new epigenetic therapy for cancer treatment. To date, all DUBs regulating ferroptosis through the inhibition of system Xc-/GPX, such as OTU DUB, ubiquitin aldehyde binding 1 (OTUB1), could directly interact with and stabilize SLC7A11 to inhibit ferroptosis, and BRCA1 associated protein 1 can decrease the amount of H2Aub occupying the SLC7A11 promoter to inhibit SLC7A11 expression, thereby inhibiting cystine uptake, resulting in increased levels of lipid peroxidation. 8,56 Moreover, it has been found that the inhibition of DUBs could be a potential anticancer therapy through the induction of GPX4 proteasomal degradation. 57 However, the number of studies on the discovery of DUBs targeting ferroptosis-related proteins is still limited.
Epigenetic regulators could regulate lipid peroxidation by affecting the expression of ferroptosis-related genes, thereby regulating the level of ferroptosis to impact the occurrence and development of tumors. 58 As an important chromatin remodeling enzyme, LSH could act as an essential inhibitor of ferroptosis in the tumorigenesis of lung cancer through interacting with WD repeat domain 76, thereby activating the lipid metabolism-related gene glucose transporter 1, the ferroptosis-related gene stearoyl-CoA desaturase 1 and fatty acid desaturases 2, and LSH can be recruited to the promoter region of SLC7A11 to enhance the transcription of SLC7A11, leading to the inhibition of ferroptosis in leukemia. 24,26 As a unique member of DUBs that could recognize unanchored polyubiquitin specifically, USP5 participates in many biological processes such as DNA repair, development, and stress response. 35,37,59 And USP5 could regulate the stability of many tumorigenesis-associated proteins, such as forkhead box M1 (FoxM1), MAF bZIP transcription factor (c-Maf), Tu translation elongation factor, mitochondrial, and snail family transcriptional repressor 2 (SNAI2) to promote the cancer progression. 42,44,60,61 However, the function of USP5 in ferroptosis is still unclear. In this study, we showed that USP5 could inhibit the ferroptosis through the stabilization of LSH. Moreover, degrasyn could reduce USP5 protein expression in a dose-dependent manner. Meantime, the treatment of degrasyn could target USP5 to increase the ubiquitin on LSH, thereby promoting the ferroptosis to suppress the HCC progression. In addition, we reveal that only USP5-WT, but not USP5-C335A, could inhibit the ferroptosis by deubiquinating LSH.
In general, we identify USP5 as a DUB of LSH that suppresses the ferroptosis of liver cancer cells to promote cell proliferation and tumor growth through stabilizing LSH protein. Our study reveals a new regulation mechanism of LSH and the effectively suppressive impact of degrasyn on HCC progression, indicating that USP5 might be further developed into a potential therapeutic target in HCC ( Figure 7F). However, the specific regulatory mechanism of LSH on SLC7A11 has not been involved in this study. Moreover, whether the inhibitory effect of USP5 on ferroptosis of cancer cells is a common phenomenon in pan-cancer remains to be further explored.

Cell culture, plasmids, sgRNAs, chemicals, and antibodies
The Cancer Research Institute of Central South University provided liver cancer cell lines HepG2, LM3, and Hep3B, normal immortal liver cells L-02, and human embryonic kidney epithelial cells 293T. In DMEM media (Gibco) with 10% fetal bovine serum (FBS), all cell lines were grown and kept at 37 • C with 5% CO 2 . Only cells that had been passed a minimum of 10 times following the first resurrection from cryopreservation and were free of mycoplasma contamination were used.
The primary antibodies utilized in this study were

Cell proliferation, migration, and colony formation assays
Cell viability was measured by CCK8 kit according to its instructions. For the migration assays, 4 × 10 4 cells with different treatments were seeded in chamber. The cells on the chamber's upper surface were removed 24 h later, and the chamber was then preserved with methanol and stained with 0.5% crystal violet. Approximately 1000 cells were planted in each well of six-well plates for the colony formation assays. Cells were methanol fixed and stained with 0.5% crystal violet after about 2 weeks.

Western blot analysis and coimmunoprecipitation (Co-IP) assay
In IP lysis buffer augmented with a protease inhibitor cocktail, the collected cells were lysed. Total protein from cell lysis was transferred to a PVDF membrane after being separated by an SDS-polyacrylamide gel. Primary antibodies were used to incubate membrane at 4 • C overnight. Bands were visualized using the ChemiDoc XRS+ image-forming system.
For immunoprecipitation, cells were collected, lysed in IP buffer with a cocktail of protease inhibitors. Complete cell lysis was precleared with 10 μL of Dynabeads Protein G (10004D, Invitrogen), and subsequently immunoprecipitated with corresponding antibodies overnight at 4 • C on a rotator. The lysates underwent an additional 2 h of incubation at 4 • C with the addition of beads. Following three cold IP buffer washes, the immunocomplexes were separated using SDS-PAGE and immunoblotted using the indicated antibodies.

RT-qPCR
TRIzol (Takara) was used to isolate the total RNA, and a PrimeScript RT reagent kit (Takara), including gDNA Eraser, was used to produce the cDNA. Utilizing the Fast-Start Universal SYBR Green Master and the ABI 7500, RT-qPCR was carried out. β-Actin was used to standardize relative gene expression levels. Table S2 includes a list of the primer sequences for RT-qPCR.

Histology and immunohistochemistry
Antigen retrieval was finished by heating tissues in sodium citrate following normal deparaffinization with xylene and hydration with ethanol of decreasing concentrations. Following that, tissues' endogenous peroxidase was blocked by incubation with 3% hydrogen peroxide. The tissues were then treated with the appropriate secondary antibody at room temperature (RT) for 30 min after being exposed to the primary antibodies, which were anti-USP5 (1:200) and anti-LSH (1:200) overnight at 4 • C. A pathologist verified the liver cancer biopsies submitted by the Department of Pathology at Xiangya Hospital (Dr. Desheng Xiao of Xiangya Hospital).

Deubiquitination assay
In 293T, HepG2, LM3, and Hep3B cells, a deubiquitination assay was conducted. HA-Ub, LSH, Flag-USP5, or USP5-C335A plasmid were transiently transfected into 293T cells. Two days after transfection, the cells were exposed to 20 μM MG132 (Selleck, S2619) for 12 h before being harvested. The cells were then immunoprecipitated to separate HA-ubiquitinated LSH. The level of LSH ubiquitination was detected using western blot. The abovementioned experimental procedures were carried out in cells stable overexpressing USP5 (HepG2, LM3) or knocking out USP5 (Hep3B).

Measurement of lipid ROS and total GSH
Cells were treated for 24 h with 6 μM RSL-3 for the lipid ROS assay, after which they were trypsinized and resuspended in DMEM media with 10% FBS and 10 μM of C11-BODIPY (D3861, Thermo Fisher). The samples were then incubated at 37 • C and 5% CO 2 for 30 min while being shielded from light. To get rid of extra C11-BODIPY, PBS was used to wash the cells twice. Dr. Shuang Liu oversaw the use of a flow cytometer (Fortessa, BD Biosciences) with fluorescein isothiocyanate green channel and Texas red channel to measure the fluorescence of C11-BODIPY581 = 591.
For the detection of Total GSH, after 24 h of incubation with 6 μM RSL-3, cells were digested and collected in centrifuge tubes, and three volumes of ice-cold 5% metaphosphoric acid were added immediately then frozen and thawed twice using liquid nitrogen and 37 • C water. The supernatant was utilized for total GSH measurement in accordance with the protocol after 10 min of centrifugation at 12,000 rpm and 4 • C.

Cell imaging of ferroptosis
In 6-well plates, 5 × 10 5 cells were planted each well. Images were captured using a microscope after 24 h of incubation with 6 μM RSL-3.

Immunofluorescence
On a 24-well plate with glass coverslips, cells were sown. The cells were frozen in methanol for 10 min at −2.0 • C after 24 h of incubation and then blocked in PBS with donkey serum (S9100, Solarbio). Cells were then incubated with primary antibodies against USP5 (10473-1-AP, Proteintech) and LSH (sc-46665, Santa Cruz Biotechnology) overnight at 4 • C, followed by an hour-long incubation at RT with Alexa Fluor 488 donkey anti-rabbit and Alexa Fluor 568 donkey anti-mouse (Life Technologies), both of which were light-protected. The nuclei were stained with DAPI (2273641, Invitrogen). A Leica fluorescence microscope was used to take the fluorescence pictures.

Nude mice and study approval
The Hunan SJA Laboratory Animal Co., Ltd. supplied the female nude mice at the age of 4 weeks. The Xiangya School of Medicine of Central South University's Institutional Animal Care and Use Committee approved the use of animals in experiments, and the experiments were carried out in accordance with the law and federal regulations for the care and protection of animals (Permit No. 2020sydw0117). 3 × 10 6 LM3/Hep3B/HepG2 cells were injected subcutaneously into the back of each mouse. As the trial progressed, tumor sizes were assessed every 3 days until the 25th day after injection for LM3 and Hep3B cells and every 2 days until the 20th day after the injection for HepG2 cells. Weighting, protein and RNA extraction, or 10% formalin fixation and paraffin embedding of tumors were all done.

Statistical analyses
All tests were carried out at least three times, with the exception of those using naked mice. Data were presented as the mean ± SD or SEM, utilizing GraphPad Prism 8.0 and Microsoft Excel 2019 for all statistical analysis. To compare two or more groups, Student's t test and the analysis of variance were used, respectively. Statistical significance was defined as a p-value <0.05.

D ATA AVA I L A B I L I T Y S TAT E M E N T
Correspondence and requests for materials or data should be addressed to Yongguang, Tao or Desheng, Xiao.

E T H I C S S TAT E M E N T
Animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of the Xiangya School of Medicine of the Central South University (Permit No. 2020sydw0117) and in compliance with the legal mandates and federal guidelines for animal protection and maintenance. All patients who donated tissues for IHC were provided with the informed consent.